A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen

A new simple immunoassay method for carcinoembryonic antigen (CEA) detection using a disposable immunosensor coupled with a flow injection system was developed. The immunosensor was prepared by coating CEA/colloid Au/chitosan membrane at a screen-printed carbon electrode (SPCE). Using a competitive immunoassay format, the immunosensor inserted in the flow system with an injection of sample and horseradish peroxidase (HRP)-labeled CEA antibody was used to trap the labeled antibody at room temperature for 35 min. The current response obtained from the labeled HRP to thionine-H(2)O(2) system decreased proportionally to the CEA concentration in the range of 0.50-25 ng/ml with a correlation coefficient of 0.9981 and a detection limit of 0.22 ng/ml (S/N=3). The immunoassay system could automatically control the incubation, washing and current measurement steps with good stability and acceptable accuracy. Thus, the proposed method proved its potential use in clinical immunoassay of CEA.     

A disposable two-throughput electrochemical immunosensor chip for simultaneous multianalyte determination of tumor markers

A disposable two-throughput immunosensor array was proposed for simultaneous electrochemical determination of tumor markers. The low-cost immunosensor array was fabricated simply using cellulose acetate membrane to co-immobilize thionine as a mediator and two kinds of antigens on two carbon electrodes of a screen-printed chip, respectively. With two simultaneous competitive immunoreactions the corresponding horseradish peroxidase (HRP) labeled antibodies were captured on the membranes, respectively, on which the immobilized thionine shuttled electrons between HRP and the electrodes for enzymatic reduction of H2O2 to produce detectable signals. The electrochemical and electronic cross-talks between the electrodes could be avoided, which was beneficial to the miniaturization of the array without considering the distance between immunosensors. Under optimal conditions the immunosensor array could be used for fast simultaneous electrochemical detection of CA 19-9 and CA 125 with the limits of detection of 0.2 and 0.4 U/ml, respectively. The serum samples from clinic were assayed with the proposed method and the results were in acceptable agreement with the reference values. The proposed method for preparation of immunosensor array could be conveniently used for fabrication of disposable electrochemical biochip with high throughput and possessed the potential of mass production and commercialization.     

Sensitive sandwich electrochemical immunosensor for alpha fetoprotein based on prussian blue modified hydroxyapatite

A sandwich electrochemical immunosensor for the sensitive determination of alpha fetoprotein (AFP) has been fabricated. Prussian blue modified hydroxyapatite (PB@HAP) was firstly prepared and used as electrochemical label due to the wonderful conductivity and good biocompatibility of HAP. The results proved that the immunosensor fabricated using the label based on PB@HAP loaded with horse radish peroxidase (HRP) and secondary anti-AFP antibody (Ab(2)) (PB@HAP-HRP-Ab(2)) had high sensitivity, and the sensitivity of the label PB@HAP-HRP-Ab(2) was much higher than labels of PB@HAP-Ab(2), PB-HRP-Ab(2) and HAP-HRP-Ab(2). The mixture of graphene sheet (GS) and thionine (TH) was not only used to immobilize anti-AFP antibody (Ab(1)) but also took part in the signal amplification. The amperometric signal increased linearly with AFP concentration in the range of 0.02-8 ng/mL with a low detection limit of 9 pg/mL. The immunosensor had the advantages of high sensitivity, good selectivity and good stability, and was applied to the analysis of AFP in serum sample with satisfactory results. Due to the low-cost and easy synthesis of PB@HAP, the screen-printed electrodes could be used instead of the bare glass carbon electrode in order to achieve mass production. In addition, it had potential application in the detection of other tumor markers.     

A novel electrochemical immunoassay based on diazotization-coupled functionalized bioconjugates as trace labels for ultrasensitive detection of carcinoembryonic antigen

In this article, a novel sandwich-type electrochemical immunosensor based on the signal amplification strategy of diazotization-coupling concept for ultrasensitive detection of carcinoembryonic antigen (CEA) was reported. It operates through physisorption of monoclonal anti-CEA on 4-aminothiophenol (4Atp) functionalized gold electrode interface as the detection platform. Diazo-4Atp-coupled-thionine (Thi)-conjugated gold nanoparticles (GNPs) were prepared for immobilization of horseradish peroxidase (HRP) and secondary anti-CEA to form core-shell bioconjugates that were used as electrochemical signal amplification reagent. The sensitivity of the immunosensor was greatly amplified by a dual amplification: one is that a large number of thionine and HRP was introduced on the electrode surface through sandwich immunoreaction, the other is that HRP as enhancer could catalyze the oxidation reaction of thionine by H(2)O(2), which results in great enhancement of the reduction peak current. Thus, the bioconjugates-based assay provided an amplification approach for detecting CEA at trace levels and led to a detection limit as low as 0.7 pg/mL (at a three times signal-to-noise ratio) that is well-below the threshold value of 2.5 ng/mL for clinical diagnosis. The assay was evaluated for clinical serum samples with various CEA concentrations and received in excellent accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA).     

A chemiluminescent immunosensor based on antibody immobilized carboxylic resin beads coupled with micro-bubble accelerated immunoreaction for fast flow-injection immunoassay

A novel immunoaffinity column used as an immunosensor for flow-injection chemiluminescent (CL) immunoassay was prepared by immobilizing antibody on carboxylic resin beads. The immunosensor could fast recognize and trap the immunocomplex of horseradish peroxidase (HRP)-labeled antibody and antigen, which was firstly formed with a micro-bubble accelerated pre-incubation process, to produce a sandwich immunocomplex. The HRP introduced in the immunoaffinity column could catalyze the CL reaction to produce enzyme-enhanced emission. With alpha-fetoprotein (AFP) as a mode, a flow-injection CL immunoassay was proposed. The whole assay for one sample, including the pre-incubation and the regeneration of immunoaffinity column, could be performed within 16min. The linear range was 1.0-80ng/ml with a correlation coefficient of 0.998 and a detection limit of 0.1ng/ml at a signal/noise ratio of 3. The intra- and inter-assay coefficients of variation at 20ng/ml AFP were 1.2% and 8.5%, respectively. The storage stability of the immunoaffinity column and the accuracy for sample detection were acceptable. This flexible, sensitive, low-cost, and rapid method is valuable for clinical immunoassay.     

Electrochemical immune-biosensor for immunoglobulin G based bioelectrocatalytic reaction on micro-comb electrodes

A new amplification strategy of electrochemical signaling from antigen-antibody interactions was proposed via back-filling immobilization of horseradish peroxidase (HRP), immunoglobulin G antibodies (anti-IgG) and gold nanoparticles onto a three-dimensional sol-gel (3DSG)-functionalized biorecognition interface. The 3DSG sol-gel network was employed not only as a building block for the surface modification but also as a matrix for ligand functionalization. The signal-amplification was based on the bioelectrocatalytic reaction of the back-filling immobilization of HRP to H(2)O(2). With the non-competitive format, the formation of the antigen-antibody complex by a simple one-step immunoreaction between the immobilized anti-IgG and IgG in sample solution inhibited partly the active center of HRP, and decreased the immobilized HRP towards H(2)O(2) reduction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to IgG in a dynamic range of 1.12-162 ng/mL with a detection limit of 0.56 ng/mL (at 3delta). Moreover, the precision, reproducibility and stability of the as-prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of biomarkers and its metastasis.     

GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein

A new flow-through electrochemical immunosensor was designed for sensitive detection of alpha-fetoprotein (AFP) in human serum by using nanogold-functionalized magnetic graphene nanosheets as immunosensing probes. Initially, amino functionalized magnetic beads were covalently immobilized on the surface of graphene oxide nanosheets (MGPs), then nanogold particles were adsorbed on the amino groups of the MGPs to construct GoldMag nanocomposites functionalized graphene nanosheets (GMGPs), and then horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP) were assembled onto the surface of nanogold particles (bio-GMGP). With the aid of an external magnet, the formed bio-GMGPs were attached onto the base electrode in the flow system. With a non-competitive immunoassay format, the injected sample containing AFP antigens was produced transparent immunoaffinity reaction with the immobilized HRP-anti-AFP on the bio-GMGPs. The formed immunocomplex inhibited partly the active center of HRP, and decreased the labeled HRP toward the reduction of H(2)O(2). The performance and factors influencing the performance of the immunosensor were investigated in detail. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.01-200 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) AFP (at 3s(B)). Intra- and inter-assay coefficients of variation (CV) were below 10%. In addition, the methodology was validated with real serum samples, receiving a good correlation with the results obtained from commercially available electrochemiluminescence automated analyzer.     

Enzyme-free electrochemical immunoassay with catalytic reduction of p-nitrophenol and recycling of p-aminophenol using gold nanoparticles-coated carbon nanotubes as nanocatalysts

A novel enzyme-free sandwich electrochemical immunoassay with an ultrahigh sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) using carbon nanotube-enriched gold nanoparticles (CNT-AuNPs) as nanolabels/nanocatalysts on anti-AFP/glutaraldehyde/thionine-modified glassy carbon electrodes (GCEs). The assays were carried out in a pH 8.0 acetic acid-buffered solution containing 6 mM p-nitrophenol (NP) and 6mM NaBH(4) after the formation of the sandwich-type immunocomplex. Initially, the NP molecules were reduced to p-aminophenol (AP) by the catalysis of the immobilized gold-nanoparticle labels on the CNT-AuNPs with the aid of NaBH(4), then the generated AP molecules were electrochemically oxidized to p-quinone imine (QI) by an electron mediator of thionine, and then the oxidized QI molecules were reduced back to APs by NaBH(4). The redox cycling of AP and QI continuously increased the signaling, leading to a high sensitivity. Compared with individual gold-nanoparticle labels, the immunosensor using CNT-AuNPs as labels displayed a wider linear range of 8.0×10(-7)-2.0×10(2) ng/mL with a lower detection limit (LOD) of 0.8 fg/mL AFP at a signal-to-noise ratio of 3, which was lower 6 orders than that of commercially available ELISA. Intra-and inter-assay coefficients of variation were below 10%. In addition, the assay was evaluated with clinical serum samples, and no significant differences at the 5% confidence level were encountered in the analysis of real samples between the proposed immunoassay and commercially available Roche 2010 Electrochemiluminescent Automatic Analyzer for determination of AFP.     

Sensitive electrochemical immunosensor for cancer biomarker with signal enhancement based on nitrodopamine-functionalized iron oxide nanoparticles

A novel electrochemical immunosensor for sensitive detection of cancer biomarker prostate specific antigen (PSA) based on nitrodopamine (NDA) functionalized iron oxide nanoparticles (NDA-Fe(3)O(4)) is described. NDA-Fe(3)O(4) was used both for the immobilization of primary anti-PSA antibody (Ab(1)) and as secondary anti-PSA antibody (Ab(2)) label. For the preparation of the label, mediator thionine (TH) was first conjugated onto NDA-Fe(3)O(4) based on the amino groups of NDA, and then the amino group of TH was used to immobilize horseradish peroxidase (HRP) and Ab(2). Due to the high amount of NDA anchored onto Fe(3)O(4) surface, the loading of antibodies as well as mediator and enzyme onto NDA-Fe(3)O(4) was substantially increased, which increased the sensitivity of the immunosensor. The resulting immunosensor displayed a wide range of linear response (0.005-50 ng/mL), low detection limit (4 pg/mL), good reproducibility and stability. The immunosensor was used to detect the PSA contents in serum samples with satisfactory results.     

Bi-enzyme synergetic catalysis to in situ generate coreactant of peroxydisulfate solution for ultrasensitive electrochemiluminescence immunoassay

A novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of α-1-fetoprotein (AFP) was designed based on the in situ bi-enzymatic reaction to generate coreactant of peroxydisulfate for signal amplification. In this work, AuNPs were electrodeposited on the glassy carbon electrode (GCE) surface, which promoted the electron transfer. Then, L-cysteine and another layer of AuNPs were, respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the ECL signal of peroxydisulfate and immobilizing antibody. At last, glucose oxidase (GOD) and horseradish peroxidase (HRP) were employed to block the nonspecific binding sites. When proper amounts of glucose were added in the detection solution, GOD catalyzed the oxidation of glucose to generate H(2)O(2), which could be further catalyzed by HRP to generate O(2) for the signal amplification. The linear range for AFP detection was 0.001-100 ng mL(-1), with a low detection limit of 3.3 × 10(-4) ng mL(-1). The novel strategy has the advantages of simplicity, sensitivity, good selectivity and reproducibility which might hold a new promise for highly sensitive bioassays applied in clinical detection.     

Label-free electrochemical immunosensor for the determination of fetoprotein based on core-shell-shell nanocomposite particles

A new approach toward the development of advanced immunosensors based on chemically functionalized core-shell-shell magnetic nanocomposite particles, and the preparation, characteristics, and measurement of relevant properties of the immunosensor useful for the detection of alpha-1-fetoprotein (AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance (APTES) (NiFe2O4@APTES) was initially prepared by covalent conjugation, then gold nanoparticles were adsorbed onto the surface of NiFe2O4@APTES, and then anti-AFP molecules were conjugated on the gold nanoparticles. The core-shell-shell nanocomposite particles not only had the properties of magnetic nanoparticles, but also provided a good biocompatibility for the immobilization of biomolecules. The core-shell-shell nanostructure present good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. The analytical performance of the immunosensor was investigated by using an electrochemical method. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of AFP, and exhibits wide linear range from 0.9 to 110 ng/mL AFP with a detection limit of 0.5 ng/mL. Moreover, the proposed immunosensors were used to analyze AFP in human serum specimens. Analytical results, obtained for the clinical serum specimen by the developed immunosensor, were in accordance with those assayed by the standard ELISA. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Simultaneous detection of NADH and H₂O₂ using flow injection analysis based on a bifunctional poly(thionine)-modified electrode

We report here a novel detection scheme for simultaneous detection of NADH and H(2)O(2) based on a bifunctional poly(thionine)-modified electrode. Electropolymerization of thionine on a "preanodized" screen-printed carbon electrode effectively lowers the oxidation potential of NADH to 0.15 V (vs. Ag/AgCl). Since poly(thionine) is also a well known electrochemical mediator for H(2)O(2) reduction, we further developed a poly(thionine)-modified ring disk electrode for simultaneous measurement of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) by flow injection analysis. By applying the optimized detection potentials of 0.2V and -0.2V at disk and ring electrodes, respectively, this system allows the simultaneous measurement of both analytes with good sensitivity (0.13 μA/mM for H(2)O(2) and 0.34 μA/mM for NADH) and limit of detection (1.74 μM and 26.0 μM for NADH and H(2)O(2)). This opens the possibility of a whole series of biosensor applications.     

Magneto-controlled electrochemical immunosensor for direct detection of squamous cell carcinoma antigen by using serum as supporting electrolyte

A new magneto-controlled microfluidic device for direct electrochemical determination of squamous cell carcinoma antigen (SCC-Ag) in serum was designed by using anti-SCC antibody (SCC-Ab)-functionalized magnetic mesoporous nanogold/thionine/NiCo(2)O(4) hybrid nanostructures as immunosensing probes (P(1)-Ab) and horseradish peroxidase-SCC-Ab conjugates-labeled nanogold/graphene nanosheets as signal tags (P(2)-Ab). In the presence of the analyte SCC-Ag, the sandwich immunocomplex was formed between the immunosensing probes and the signal tags. With the aid of an external magnet, the formed immunocomplex was attached to the microfluidic device. The assay was implemented in newborn calf serum (NBCS) containing 2.5 mM H(2)O(2) based on the labeled peroxidase on the P(2)-Ab toward the catalytic reduction of H(2)O(2). Under optimal conditions, the increase in the current was proportional to the concentration of SCC-Ag from 2.5 pg/mL to 15 ng/mL. The detection limit (LOD) was 1.0 pg/mL SCC-Ag at 3s(B). The electrochemical immunoassay displayed an acceptable precision, selectivity and stability. Clinical serum specimens were assayed with the method, and the results were in acceptable agreement with those obtained from the referenced electrochemiluminescent method. Importantly, the method can be suitable for on-line use in the mass production of miniaturized lab-on-a-chip devices and open a new opportunity for protein diagnostics and biosecurity.     

Electrochemical DNA base pairs quantification and endonuclease cleavage detection

A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.     

An organic–inorganic hybrid nanostructure-functionalized electrode for electrochemical immunoassay of biomarker by using magnetic bionanolabels

A new electrochemical immunoassay of alpha-fetoprotein (AFP) was developed on an organic–inorganic hybrid nanostructure-functionalized carbon electrode by coupling with magnetic bionanolabels. Multi-walled carbon nanotubes (CNTs), single-stranded DNA, thionine and AFP were utilized for the construction of the immunosensor, while the core–shell Fe₃O₄-silver nanocomposites were employed for the label of horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-AgFe). Electrochemical measurement toward AFP was carried out by using magnetic bionanolabels as traces and H₂O₂ as enzyme substrate with a competitive-type immunoassay mode. Experimental results indicated that the immunosensors with carbon nanotubes and DNA exhibited better electrochemical responses than those of without carbon nanotubes or DNA. Under optimal conditions, the electrochemical immunosensor by using HRP-anti-AFP-AgFe as signal antibodies exhibited a linear range of 0.001–200 ng mL⁻¹ AFP with a low detection limit of 0.5 pg mL⁻¹ at 3s_B. Both intra- and inter-assay coefficients of variation were 7.3%, 9.4%, 8.7% and 10.2%, 7.8%, 9.4% toward 0.01, 30, 120 ng mL⁻¹ AFP, respectively. The specificity and stability of the electrochemical immunoassay were acceptable. In addition, the methodology was validated for 12 clinical serum specimens including 9 positive specimens and 3 normal specimens, receiving a good correlation with the results obtained from the referenced electrochemiluminescence assay.     

A new homogeneous enzyme immunoassay. Its application to measurement of alpha-fetoprotein

A rapid and sensitive homogeneous enzyme immunoassay (homogeneous EIA) was developed for determination of serum proteins such as alpha-fetoprotein (AFP). There are two assay systems, one is a competitive system including horseradish peroxidase (HRP)-labeled antigen, antibody and substrate, and the other is a non-competitive system including HRP-labeled antibody and substrate. When the aggregate was formed through the binding of HRP-labeled AFP and anti-AFP antibody or through the binding of HRP-labeled anti-AFP antibody and AFP, HRP of the aggregates, as compared with HRP of free conjugates, exhibited marked activity in the presence of 35 mM H2O2. The extent of stimulation of HRP activity depended on the amount of AFP. This new assay method is very simple and sensitive, and can be used for the determination of any kind of protein, hormone, or drug.     

A disposable electrochemical immunosensor based on carbon screen-printed electrodes for the detection of prostate specific antigen

A novel screen-printed electrode (SPEs) on sheets of vegetable parchment was prepared. The obtained SPEs were stable, convenient, inexpensive and suitable for large-area screen-printing. With these SPEs, we explored the fabrication of a novel, disposable and highly sensitive electro-analytical immunosensor using graphene nanosheets (GS) and horseradish peroxidase (HRP)-labeled signal antibody functionalized with gold nanoparticles (HRP-Ab(2)/Au NPs). GS was used to increase the conductivity and stability of this immunosensor due to its fast electron transportation and good biocompatibility. Au NPs could not only provide a large surface area for the immobilization of HRP-Ab(2) but also enhance the electroreduction between HRP and H(2)O(2) to amplify the electrochemical signal on the sandwich immuno-complexes modified SPEs. The proposed SPEs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical methods involving cyclic voltammetry (CV), and electrochemical impedence method. Using prostate specific antigen (PSA) as a model analyte, this immunosensor showed a wide linear range over 6 orders of magnitude with the minimum value down to 2pgmL(-1). In addition, this immunosensor could avoid the need of deoxygenation for the electrochemical immunoassay. Thus, it provided a promising potential in clinical applications.     

Electrochemical immunoassay for carcinoembryonic antigen based on signal amplification strategy of nanotubular mesoporous PdCu alloy

Interests in using nanoporous metals for biosensing applications have been increasing. Herein, nanotubular mesoporous PdCu (NM-PdCu) alloy is used to fabricate a novel label-free electrochemical immunosensor for cancer biomarker carcinoembryonic antigen (CEA). It operates through physisorption of anti-CEA on NM-PdCu and the mixture of sulfonated graphene sheets (HSO(3)-GS) and thionine (TH) functionalized glassy carbon electrode interface as the detection platform. In this study, chitosan (CS)-PdCu is bound very strongly to carcinoembryonic antibody (anti-CEA), because of the good electron conductivity, high surface area, and good biocompatibility. CS-PdCu is immobilized on electrodes by electrostatic interactions between the negatively charged sulfo group of HSO(3)-GS and the abundant positively charged amino groups of chitosan. TH acts as the redox probe. Under the optimized conditions, the electrochemical immunosensor exhibits a wide working range from 0.01 to 12 ng/mL with a low detection limit of 4.86 pg/mL. The accuracy, reproducibility, and stability of the immunosensor are acceptable. The assay is evaluated for real serum samples, receiving satisfactory results. The nanoporous metal materials-based immunoassay provides a promising approach in clinical application and thus represents a versatile detection method.     

Multifunctional Fe₃O₄ core/Ni-Al layered double hydroxides shell nanospheres as labels for ultrasensitive electrochemical immunoassay of subgroup J of avian leukosis virus

A novel electrochemical immunosensor for ultrasensitive detection of subgroup J of avian leukosis virus (ALVs-J) was designed by using graphene sheets (GS)-layered double hydroxides (LDHs) composites modified electrode with multifunctional Fe(3)O(4) core/Ni-Al LDHs shell (LDHs@Fe(3)O(4)) nanospheres as labels. At first, the GS-LDHs were used for the immunosensor platform for improving the electronic transmission rate as well as increasing the surface area to capture a large amount of primary antibodies (Ab(1)). After that, ferrocene (Fc), secondary antibodies (Ab(2)) and horseradish peroxidase (HRP) multifunctional LDHs@Fe(3)O(4) nanospheres were used as labels with high load amount and good biological activity. Subsequently, in presence of H(2)O(2), amplified signals were obtained by an electrochemical sandwich immunoassay protocol. To embody the signal amplification property of the protocol, the analytical properties of various immunosensor platform and labels were compared in detail. Under optimal conditions, the reduction peak currents of the electrochemical immunosensor were proportional to the ALVs-J concentration over the range from 10(2.32) to 10(5.50) TCID(50)/mL with a low detection limit (180 TCID(50)/mL, S/N=3). The resulting immunosensor also displayed a good selectivity, reproducibility and stability.     

Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybrids platform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a model protein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels and secondary antibodies (Ab₂) linked to GNPs at high HRP/Ab₂ molar ratio. The approach provided a linear response range between 0.125 and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.